WO2006078618A2 - Compositions for use as a signal generation component and methods of using same - Google Patents
Compositions for use as a signal generation component and methods of using same Download PDFInfo
- Publication number
- WO2006078618A2 WO2006078618A2 PCT/US2006/001529 US2006001529W WO2006078618A2 WO 2006078618 A2 WO2006078618 A2 WO 2006078618A2 US 2006001529 W US2006001529 W US 2006001529W WO 2006078618 A2 WO2006078618 A2 WO 2006078618A2
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- composition
- carrier
- aminodextran
- formula
- metal chelate
- Prior art date
Links
- 239000000203 mixture Substances 0.000 title claims abstract description 49
- 230000007274 generation of a signal involved in cell-cell signaling Effects 0.000 title claims abstract description 18
- 238000000034 method Methods 0.000 title claims abstract description 18
- 229910052751 metal Inorganic materials 0.000 claims abstract description 47
- 239000002184 metal Substances 0.000 claims abstract description 47
- 239000003446 ligand Substances 0.000 claims abstract description 29
- 239000013522 chelant Substances 0.000 claims abstract description 28
- 238000000576 coating method Methods 0.000 claims abstract description 26
- 239000011248 coating agent Substances 0.000 claims abstract description 25
- 238000003018 immunoassay Methods 0.000 claims abstract description 16
- 229910052693 Europium Inorganic materials 0.000 claims abstract description 14
- OGPBJKLSAFTDLK-UHFFFAOYSA-N europium atom Chemical compound [Eu] OGPBJKLSAFTDLK-UHFFFAOYSA-N 0.000 claims abstract description 14
- FIQMHBFVRAXMOP-UHFFFAOYSA-N triphenylphosphane oxide Chemical compound C=1C=CC=CC=1P(C=1C=CC=CC=1)(=O)C1=CC=CC=C1 FIQMHBFVRAXMOP-UHFFFAOYSA-N 0.000 claims abstract description 6
- 229910052772 Samarium Inorganic materials 0.000 claims abstract description 5
- 229910052771 Terbium Inorganic materials 0.000 claims abstract description 5
- KZUNJOHGWZRPMI-UHFFFAOYSA-N samarium atom Chemical compound [Sm] KZUNJOHGWZRPMI-UHFFFAOYSA-N 0.000 claims abstract description 5
- GZCRRIHWUXGPOV-UHFFFAOYSA-N terbium atom Chemical compound [Tb] GZCRRIHWUXGPOV-UHFFFAOYSA-N 0.000 claims abstract description 5
- 229910052692 Dysprosium Inorganic materials 0.000 claims abstract description 4
- KJTLSVCANCCWHF-UHFFFAOYSA-N Ruthenium Chemical compound [Ru] KJTLSVCANCCWHF-UHFFFAOYSA-N 0.000 claims abstract description 4
- KBQHZAAAGSGFKK-UHFFFAOYSA-N dysprosium atom Chemical compound [Dy] KBQHZAAAGSGFKK-UHFFFAOYSA-N 0.000 claims abstract description 4
- 229910052762 osmium Inorganic materials 0.000 claims abstract description 4
- SYQBFIAQOQZEGI-UHFFFAOYSA-N osmium atom Chemical compound [Os] SYQBFIAQOQZEGI-UHFFFAOYSA-N 0.000 claims abstract description 4
- 229910052707 ruthenium Inorganic materials 0.000 claims abstract description 4
- 238000012935 Averaging Methods 0.000 claims abstract 2
- 239000002245 particle Substances 0.000 claims description 26
- 239000012491 analyte Substances 0.000 claims description 16
- 239000004816 latex Substances 0.000 claims description 11
- 229920000126 latex Polymers 0.000 claims description 11
- -1 dextran aldehyde Chemical class 0.000 claims description 9
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 7
- 229910052760 oxygen Inorganic materials 0.000 claims description 7
- 239000001301 oxygen Substances 0.000 claims description 7
- 125000001424 substituent group Chemical group 0.000 claims description 6
- 229920002307 Dextran Polymers 0.000 claims description 5
- 125000003118 aryl group Chemical group 0.000 claims description 2
- 230000005465 channeling Effects 0.000 claims description 2
- 125000005265 dialkylamine group Chemical group 0.000 claims description 2
- 150000002170 ethers Chemical class 0.000 claims description 2
- 125000004051 hexyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])* 0.000 claims description 2
- 238000012360 testing method Methods 0.000 claims description 2
- 150000003568 thioethers Chemical class 0.000 claims description 2
- 239000011324 bead Substances 0.000 description 23
- 239000000975 dye Substances 0.000 description 21
- 230000004044 response Effects 0.000 description 12
- 230000009870 specific binding Effects 0.000 description 9
- 150000001875 compounds Chemical class 0.000 description 8
- 239000000463 material Substances 0.000 description 8
- PPBRXRYQALVLMV-UHFFFAOYSA-N Styrene Natural products C=CC1=CC=CC=C1 PPBRXRYQALVLMV-UHFFFAOYSA-N 0.000 description 7
- 230000027455 binding Effects 0.000 description 7
- 230000009871 nonspecific binding Effects 0.000 description 7
- MYMOFIZGZYHOMD-UHFFFAOYSA-N Dioxygen Chemical compound O=O MYMOFIZGZYHOMD-UHFFFAOYSA-N 0.000 description 6
- 239000007987 MES buffer Substances 0.000 description 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 239000000969 carrier Substances 0.000 description 6
- 239000004793 Polystyrene Substances 0.000 description 5
- 239000003153 chemical reaction reagent Substances 0.000 description 5
- 229920000642 polymer Polymers 0.000 description 5
- 229920002223 polystyrene Polymers 0.000 description 5
- 238000000159 protein binding assay Methods 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- RNGCGRQNGQFBJS-UHFFFAOYSA-N 4,4,5,5,6,6,6-heptafluoro-1-[4-[2-[4-(4,4,5,5,6,6,6-heptafluoro-3-oxohexanoyl)phenyl]phenyl]phenyl]hexane-1,3-dione Chemical compound C1=CC(C(=O)CC(=O)C(F)(F)C(F)(F)C(F)(F)F)=CC=C1C1=CC=CC=C1C1=CC=C(C(=O)CC(=O)C(F)(F)C(F)(F)C(F)(F)F)C=C1 RNGCGRQNGQFBJS-UHFFFAOYSA-N 0.000 description 4
- 239000000370 acceptor Substances 0.000 description 4
- 230000005281 excited state Effects 0.000 description 4
- ZMBHCYHQLYEYDV-UHFFFAOYSA-N trioctylphosphine oxide Chemical compound CCCCCCCCP(=O)(CCCCCCCC)CCCCCCCC ZMBHCYHQLYEYDV-UHFFFAOYSA-N 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 125000003277 amino group Chemical group 0.000 description 3
- 238000003556 assay Methods 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 230000000052 comparative effect Effects 0.000 description 3
- 229920001577 copolymer Polymers 0.000 description 3
- 150000004676 glycans Chemical class 0.000 description 3
- 229920001282 polysaccharide Polymers 0.000 description 3
- 239000005017 polysaccharide Substances 0.000 description 3
- 238000001179 sorption measurement Methods 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide Chemical compound CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 description 2
- ARXJGSRGQADJSQ-UHFFFAOYSA-N 1-methoxypropan-2-ol Chemical compound COCC(C)O ARXJGSRGQADJSQ-UHFFFAOYSA-N 0.000 description 2
- KAKZBPTYRLMSJV-UHFFFAOYSA-N Butadiene Chemical compound C=CC=C KAKZBPTYRLMSJV-UHFFFAOYSA-N 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- 239000004698 Polyethylene Substances 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 230000006978 adaptation Effects 0.000 description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 2
- 125000000524 functional group Chemical group 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 239000000017 hydrogel Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 229920002401 polyacrylamide Polymers 0.000 description 2
- 229920000573 polyethylene Polymers 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 229920002554 vinyl polymer Polymers 0.000 description 2
- 229920000936 Agarose Polymers 0.000 description 1
- CVBUKMMMRLOKQR-UHFFFAOYSA-N CC(CC(c1ccccc1)=O)=O Chemical compound CC(CC(c1ccccc1)=O)=O CVBUKMMMRLOKQR-UHFFFAOYSA-N 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 229910020889 NaBH3 Inorganic materials 0.000 description 1
- 239000000020 Nitrocellulose Substances 0.000 description 1
- 239000004677 Nylon Substances 0.000 description 1
- 229920002845 Poly(methacrylic acid) Polymers 0.000 description 1
- 239000004743 Polypropylene Substances 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- WRVRNZNDLRUXSW-UHFFFAOYSA-N acetic acid;prop-2-enoic acid Chemical compound CC(O)=O.OC(=O)C=C WRVRNZNDLRUXSW-UHFFFAOYSA-N 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- PNEYBMLMFCGWSK-UHFFFAOYSA-N aluminium oxide Inorganic materials [O-2].[O-2].[O-2].[Al+3].[Al+3] PNEYBMLMFCGWSK-UHFFFAOYSA-N 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- RJGDLRCDCYRQOQ-UHFFFAOYSA-N anthrone Chemical compound C1=CC=C2C(=O)C3=CC=CC=C3CC2=C1 RJGDLRCDCYRQOQ-UHFFFAOYSA-N 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 238000002820 assay format Methods 0.000 description 1
- 230000000712 assembly Effects 0.000 description 1
- 238000000429 assembly Methods 0.000 description 1
- 239000005312 bioglass Substances 0.000 description 1
- NTXGQCSETZTARF-UHFFFAOYSA-N buta-1,3-diene;prop-2-enenitrile Chemical compound C=CC=C.C=CC#N NTXGQCSETZTARF-UHFFFAOYSA-N 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002301 cellulose acetate Polymers 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000000919 ceramic Substances 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000004737 colorimetric analysis Methods 0.000 description 1
- 230000009918 complex formation Effects 0.000 description 1
- 230000001010 compromised effect Effects 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 229920006037 cross link polymer Polymers 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000011026 diafiltration Methods 0.000 description 1
- 150000001993 dienes Chemical class 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 125000001475 halogen functional group Chemical group 0.000 description 1
- 229910001385 heavy metal Inorganic materials 0.000 description 1
- 239000002198 insoluble material Substances 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 239000000696 magnetic material Substances 0.000 description 1
- 239000006249 magnetic particle Substances 0.000 description 1
- 229910001510 metal chloride Inorganic materials 0.000 description 1
- 150000002739 metals Chemical class 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 229920001220 nitrocellulos Polymers 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 229920001778 nylon Polymers 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 125000005010 perfluoroalkyl group Chemical group 0.000 description 1
- 150000003904 phospholipids Chemical class 0.000 description 1
- 239000003504 photosensitizing agent Substances 0.000 description 1
- 229920000058 polyacrylate Polymers 0.000 description 1
- 239000004584 polyacrylic acid Substances 0.000 description 1
- 229920000139 polyethylene terephthalate Polymers 0.000 description 1
- 239000005020 polyethylene terephthalate Substances 0.000 description 1
- 229920000193 polymethacrylate Polymers 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 229920001155 polypropylene Polymers 0.000 description 1
- 229920000915 polyvinyl chloride Polymers 0.000 description 1
- 239000004800 polyvinyl chloride Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 239000010420 shell particle Substances 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- 239000011856 silicon-based particle Substances 0.000 description 1
- 229910052709 silver Inorganic materials 0.000 description 1
- 239000004332 silver Substances 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 238000000527 sonication Methods 0.000 description 1
- 150000003440 styrenes Chemical class 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
- G01N33/586—Liposomes, microcapsules or cells
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2458/00—Labels used in chemical analysis of biological material
- G01N2458/40—Rare earth chelates
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/968—High energy substrates, e.g. fluorescent, chemiluminescent, radioactive
Definitions
- the present invention relates generally to compositions useful as signal generation components in immunoassays.
- the invention relates to compositions that simultaneously provide a strong signal as well as an enhanced specificity for the analytes of interest.
- Immunoassay reagents have functionality that can be broken down into two broad components: signal generation (also known as amplification) functionality, and ligand binding functionality.
- signal generation functionality is required for detection of ligand binding to analyte
- ligand binding functionality is the specificity of the reagent for the analyte.
- the ligand binding activity is accomplished by covalently attaching ligands to the particle surface.
- Antibodies and small haptens of biological significance, such as thyroxin, are examples of common ligands.
- Flourescent and chemiluminescent compositions have been widely used in signal generation components of immunoassays.
- a typical signal generation component includes a carrier, such as a latex particle, dyed with a flurosecent or chemiluminescent composition.
- metal chelates have been widely used as fluroscent and chemiluminescent dyes because of their generally large stokes' shift, sharp emission peak and long emission wavelength.
- the metal chelates are a complex formed of a metal, such as europium, samarium, or terbium, and ligands such as thiophenetrifluorobutanedione (TTA), napthyltriflurobutanedione (NTA), and 4, 7- diphenyl-1 ,10-phenathroline (DPP) trioctyl phosphine oxide (TOPO), triphenyl phosphine oxide (TPPO).
- TTA thiophenetrifluorobutanedione
- NDA napthyltriflurobutanedione
- DPP 4, 7- diphenyl-1 ,10-phenathroline
- TOPO trioctyl phosphine oxide
- TPPO triphenyl phosphine oxide
- Some commonly used metal chelates include Eu(TTA) 3 DPP and Eu(NTA) 3 DPP.
- Dyed carriers that are used to make assay reagents must have both the capability to provide signal generation, and the chemical functionality for covalent attachment of ligands.
- the process used to attach the ligands can be critical to the quality of the specificity of the ligand binding functionality.
- the specificity of the ligands can be compromised in several ways by inappropriate choices of attachment chemistry. For example, if passive adsorption of the ligands occurs simultaneously with covalent attachment, the passively adsorbed ligands may come off of the carrier during the assay. The resulting free ligand will interfere with the assay and reduce its sensitivity. Another problem that can occur is the non-specific binding of the reagent to the other carrier, which leads to elevation of the immunoassay signal in the absence of analyte.
- Ligands are often attached to polystyrene particles through carboxy groups that are attached directly to the surface of the particles. This approach can have both of the problems described above.
- the passive adsorption and nonspecific binding problems can be avoided by introducing one or more layers of immobilized hydrogel polymers, such as an aminodextran, between the polystyrene particles and the ligands. It is important for elimination of adsorption and nonspecific binding to have a continuous layer of these hydrogel polymers.
- the coating of particles that are dyed with conventional dyes such as Eu(TTA) 3 DPP or Eu(NTA) 3 DPP typically result in coating densities of aminodextran that are significantly less than optimal for preventing non-specific binding.
- the coupling density of the aminodextran is typically reduced by 40-50% from its maximum when these dyes are added at the optimum level for the chemiluminescent response of the reagent.
- Lower levels of dye give compositions that interfere less with the aminodextran coating process, but reducing the dye content also reduces the chemiluminescence necessary for a strong signal.
- compositions of the present invention provide signal generation components having an optimal balance between the reduction of non-specific binding while allowing an improved response signal.
- a composition suitable for use as a signal generation component in an immunoassay includes a carrier having a coating of an aminodextran and dyed with a metal chelate.
- the metal chelate is present in the amount of at least 0.065 ⁇ M per milligram of carrier, and the average aminodextran coating density is at least about 45 ⁇ g per milligram of carrier.
- a composition according to another aspect of the invention includes a carrier coated with an aminodextran and with a complex incorporated therein having the formula: wherein M is a metal selected from the group consisting of europium, terbium, dysprosium, samarium, osmium and ruthenium;
- L1 is a ligand selected from the group consisting of DPP, TOPO, and TPPO; L2 comprises a ligand having the formula
- a method of detecting the presence or amount of an analyte in a test sample suspected of containing the analyte comprises performing an immunoassay using the compositions of the present invention as a signal generating component.
- FIG. 1 is a schematic illustrating the synthesis of one embodiment of a metal chelate according to the present invention.
- FIG. 2 is a schematic illustrating the synthesis of another embodiment of a metal chelate according to the present invention.
- FIG. 3 is a graphical representation of aminodextran concentration and response signal as functions of metal concentration for a prior art composition.
- FIG. 4 is a graphical representation of aminodextran concentration and response signal as functions of metal concentration for compositions according to the present invention.
- compositions of the present invention are useful as signal generation components in immunoassays.
- the compositions generally include a carrier coated with an aminodextran and have a metal chelate incorporated therein.
- the compositions provide strong response signals which allows for quantitative determinations of analytes of interest, while reducing errors caused by non-specific binding.
- Carriers suitable for the present invention include solid phase materials, typically a support or surface, that can have any one of a number of shapes, such as strip, sheet, rod, plate, well, tube, particle or bead.
- the material is usually of an organic or inorganic, swellable or non-swellable, porous or non-porous, magnetic or nonmagnetic, water-insoluble material.
- the surface can be hydrophilic or capable of being rendered hydrophilic.
- the solid support includes inorganic powders such as silica, magnesium sulfate, and alumina; natural polymeric materials, particularly cellulosic materials and materials derived from cellulose, such as fiber containing papers, e.g., filter paper, chromatographic paper, etc.; synthetic or modified naturally occurring polymers, such as nitrocellulose, cellulose acetate, polyvinylchloride, polyacrylamide, crosslinked dextran, agarose, polyacrylate, polyethylene, polypropylene, poly(4- methylbutene), polystyrene, polymethacrylate, polyethyleneterephthalate, nylon, polyvinylbutyrate, etc.; either used by themselves or in conjunction with other materials; glass available as Bioglass, ceramics, magnetic materials, metals, and the like. Natural or synthetic assemblies such as liposomes, phospholipid vesicles, and cells can also be employed.
- the carrier is a particle.
- Particle encompasses spheres, spheroids, beads and other shapes as well. Suitable particles are typically at least 20 nm and not more than about 20 ⁇ m, usually at least about 40 nm and less than 10 ⁇ m, preferably 0.1 to 10 ⁇ m, more preferably, 0.1 to 5 ⁇ m, and even more preferably 0.15 to 3 ⁇ m.
- the particle may be organic or inorganic, swellable or non-swellable, porous or non-porous, having any density, but preferably of a density approximating water, generally from about 0.7 to about 1.5 g/ml, preferably suspendible in water, and composed of material that can be transparent, partially transparent, or opaque.
- the particles may be core-and-shell particles, such as particles with a magnetic core and a hard shell coating of polymerized monomer(s).
- the particles are preferably negative charged.
- the particles are preferably solid, e.g., polymer particles, metal sols (particles comprised of a heavy metal such as, e.g., gold or silver), glass particles, silicon particles, magnetic particles, dye crystallites.
- Latex particle means a particulate water-suspendible water-insoluble polymeric material.
- the latex is frequently a substituted polyethylene such as: polystyrene-butadiene, polyacrylamide polystyrene, polystyrene with amino groups, poly-acrylic acid, polymethacrylic acid, acrylonitrile-butadiene, styrene copolymers, polyvinyl acetate- acrylate, polyvinyl pyrridine, vinyl-chloride acrylate copolymers, and the like.
- Non- crosslinked polymers of styrene and carboxylated styrene or styrene functionalized with other active groups such as amino, hydroxyl, halo and the like are preferred. Frequently, copolymers of substituted styrenes with dienes such as butadiene will be used.
- the carriers according to the present invention are coated with one or more layers layers of an aminodextran.
- An aminodextran is a derivatized glucose polymer with amino groups having a molecular weight of about 10,000 to about 2,000,000, preferably about 500,000.
- Methods of preparing aminodextrans and coating carriers are known in the art. Suitable methods are described in US 5,707,877, columns 18-20, and US 5,639,620, columns 1 21 and 22.
- aminodextran Is coated on the carrier by covalent attachment of the amino group of aminodextran to the surface carboxyl groups or other amine-reactive functional group of the carrier.
- Such a method is described in provided in commonly-assigned and copending US Ser. No. 10/220,623, filed March 6, 2000 entitled “Carriers Coated with Polysaccharides, Their Preparation and Use,” the entire disclosure of which is incorporated herein by reference.
- the aminodextran coating improves the specificity of the carrier in specific binding assays and therefore improves the sensitivity of immunoassays. It has been discovered that the metal chelate of the present invention unexpectedly allows for coating of carriers with aminodextran in the amount of at least about 45 ⁇ g per milligram of carrier in the presence of a dye concentration as high as at least 0.065 ⁇ M. According to one preferred embodiment of the present invention, the aminodextran coating is present in at least about 49 ⁇ g per milligram of carrier.
- the carrier may be coated with a second layer comprising a polysaccharide, as described in U.S. Ser. No 10/220,623.
- the polysaccharide may be covalently coupled to the aminodextran layer by amine-reactive functional groups.
- the second layer comprises dextran aldheyde.
- a metal chelate is incorporated into the carrier through a known process, such as dyeing.
- the metal chelate allows for a fluorescent or chemiluminescent signal to be detected.
- the metal chelate is chosen such that relatively higher concentrations of the metal chelate do not interfere with the binding of the aminodextran coating to the carrier.
- the metal chelate is present in the amount of at least 0.065 ⁇ M per milligram of carrier, while the aminodextran average coating density is at least about 45 ⁇ g per milligram of carrier.
- the metal chelate is present in the amount of at least 0.065 ⁇ M and less than about 0.150 ⁇ M per milligram of carrier, more preferably about 0.079 ⁇ M to about 0.150 ⁇ M per milligram of carrier, and more preferably about 0.087 ⁇ M per milligram of carrier. In another embodiment of the invention, the metal chelate is present in the amount of about 0.110 ⁇ M to about 135 ⁇ M. These concentrations of metal chelate and aminodextran have been found to provide a proper balance of response signal and component specificity for the analyte of interest.
- the metal chelate has the general formula wherein M is a metal selected from the group consisting of europium, terbium, dysprosium, samarium, osmium and ruthenium;
- L1 is a ligand selected from the group consisting of DPP, TOPO, and TPPO; L2 comprises a ligand having the formula
- Suitable electron donating groups are those that make available extra negative charge to the perfluoralkyl groups.
- the electron donating group R is selected from the group consisting of Ci to C 6 alkyls, dialkylamines, ethers, thioethers, and aryl groups.
- the metal chelates of the present invention are effective in allowing optimized amounts of amindoextran to coat the carrier because the metal chelates provide an unexpectedly stable complex relative to prior metal chelate dyes.
- the stability of the complex reduces the amount of the free metal, which is believed to inhibit the attachment of the aminodextran to the carrier.
- electron donating groups R further add to the stability of the complex by making available additional negative charge to the perfluoroalkyl groups of the ligands, thereby improving the ability of the perfluoralkyl group to bind to the metal in the complex.
- M is europium and the carrier is a latex particle.
- L2 is selected from among the following formulas:
- the ligands L2 are 2-(1',1',I 1 , 2", 2',3 ⁇ 3'- heptafluoro-4 1 , 6'-hexanedion-6'-yl)-naphthylene (NHA) and 4, 4'-bis (1",1",1",2", 2", 3", 3"- heptafluroro- 4", 6" - hexanedion-6"-yl)-o-terphenyl (BHHT).
- the metal chelate comprises Eu(NHA) 3 DPP and Eu(BHHT) 2 DPP.
- the metal chelates can be prepared by combining the metal chloride with the desired ratio of metal ligand molecules in an organic solvent and sufficient base to take up the released hydrochloric acid. Suitable syntheses are illustrated in FIGS. 1-2, respectively.
- the metal chelate dyes of the present invention have been found to allow for a higher density of aminodextran coating at relatively higher concentrations of the metal chelate dyes when compared to conventional dyes, as demonstrated in the Comparative Examples below.
- the metal chelates of the present invention generally will allow an aminodextran coating at about 45 to about 55 ⁇ g per mg beads compared to about 23 to about 35 ⁇ g per mg of bead for the conventional dyes.
- the dyes of the present invention can be present in amounts sufficient to give strong response signals as part of a signal generation component in a specific binding assay, while allowing the carrier to be coated with an aminodextran in an amount sufficient to reduce the amount of non- specific binding.
- compositions of the present invention are suitable for use as a signal generation component in specific binding assays.
- the compositions are suitable for any immunoassay in which a signal is measured in response to ligand binding to an analyte.
- Those skilled in the art will readily appreciate the compositions have wide utility in a number of assay formats.
- the signal generating component is used to perform Luminescent Oxygen Channeling Immunoassay (LOCITM) as described in EP-A2-0 515 194.
- LOCITM Luminescent Oxygen Channeling Immunoassay
- at least one specific binding partner is bound to a carrier according to the present invention.
- a medium suspected of containing an analyte is treated under conditions such the analyte affects the amount of a sensitizer capable in its excited state of generating singlet oxygen and a chemiluminescent compound that can come into close proximity such that singlet oxygen generated by said photosensitizer can activate said chemiluminescent compound, which subsequently produces light, and measuring the light, the amount thereof being related to the amount of analyte in said medium.
- the LOCITM method includes the steps of: (A) combining either simultaneously or wholly or partially sequentially (i) a medium suspected of containing the analyte; (ii) a first specific binding partner associated with a sensitizer capable in its excited state of generating singlet oxygen; and (iii) a second specific binding partner associated with a composition comprising a chemiluminescent compound, which is a substance that undergoes a chemical reaction with singlet oxygen to form a metastabile intermediate species that can decompose with the simultaneous or subsequent emission of light; (B) allowing the formation of complexes comprising the first and the second specific binding partner, the complex formation brings the sensitizer into close proximity to the chemiluminescent compound, (C) activating the sensitizer to generate singlet oxygen; and measuring the amount of light emitted by the chemiluminescent compound, the light is directly or inversely proportional to the amount of analyte in the medium.
- the composition comprising the chemiluminescent compound can also comprise one or more fluorescent molecules which are excited by the activated chemiluminescent compound.
- the light emitted by said fluorescent molecules can be measured to determine the amount of analyte in the medium.
- the carrier may be dyed both with a metal chelate and an oxygen acceptor.
- the oxygen acceptor is a component that reacts with singlet oxygen to form a highly unstable product that rapidly decomposes into an excited state. The excess energy of the excited state is transferred to the dye by a Forster energy transfer that takes place in a few nanoseconds.
- the metal chelate dye then emits a photon of light that can be detected with a photon counter.
- Suitable oxygen acceptors are disclosed in U.S. Pat. Nos. 5,578,498 and 5,811,311.
- One particularly preferred oxygen acceptor is C-28 thioxene, which has the following formula:
- the aminodextran coating is then applied as follows. 1mL of the dyed beads ( ⁇ 20mg/ml) is mixed with 20mg/ml of hydroxypropylaminodextran (MW 500K) in 0.05M MES / pH 6.0 in the presence of 3.8 mg/ml EDAC. After incubating this mixture for 16 hours at room temperature, (in the dark) the beads were washed once with 2ml of 0.05M MES / pH 6.0, then with 6ml of 0.05M MES, 1.0M NaCl/ pH 6.0. Finally the beads were resuspended in 1 ml of 0.05M MES / pH 6.0 to yield the coated beads. Washing was performed by centrifugation method (using Sorva) RC-5B Plus centifruge or Ependorf centrifuge-5415 C) and pellets were resuspended by sonication (using Branson Sonifer-450).
- the dextran aldehyde layer may then be added as follows. 1ml of the
- a first set of latex particles were dyed with Eu (NTA) 3 DPP and thioxene and coated with an aminodextran according to the methods described herein.
- a second set of latex particles were dyed with Eu (NHA) 3 DPP and thioxene and coated with an aminodextran.
- the aminodextran coating densities and the chemiluminesence were simultaneously measured at several concentrations of the metal chelate.
- the aminodextran coating density was measured by a colorimetric test after being treated with anthrone and 80% sulfuric acid.
- the chemiluminescence was measured as LOCI counts by a Envision-Alpha reader sold by Perkin-Elmer.
- the data points for Eu (NTA) 3 DPP are tabulated in Table 1 and shown graphically with curve fitting in FIG. 3.
- the data points for Eu(NHA) 3 DPP are tabulated in Table 2 and, for concentrations up to 0.094, are shown graphically with curve fitting in FIG. 4.
- Table 1 LOCI counts and aminodextran concentration as a function of europium concentration in E (NTA ⁇ DPP
- Table 2 LOCI counts and aminodextran concentration as a function of europium concentration in Eu (BHHT) 2 DPP
- the compound according to the present invention begins to plateau both in terms of aminodextran concentration and LOCI signal with as little as 0.065um Eu / gram of beads.
- the average aminodextran coating concentration is 49 ⁇ g/mg beads.
- the concentration of aminodextran generally holds steady as the concentration of europium is increased.
- Latex beads from Seradyn were dyed with 0.171 mmo! of Europium dye and 0.303 mmol of C-28 thioxene per gram of bead for each of two conventional dyes, Eu(TTA) 3 DPP and Eu(NTA) 3 DPP and two dyes according to the present invention, Eu(BHHT) 2 DPP and Eu(NHA) 3 DPP.
- the beads were then coated with aminodextran as described herein. The densities of the aminodextran was then determined, as well as the amplitude of its response signal. The data are tabulated on Table 3.
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ES06718583T ES2392730T3 (es) | 2005-01-21 | 2006-01-17 | Composiciones para el uso como un componente de generación de señales y métodos par usar las mismas |
JP2007552204A JP4741607B2 (ja) | 2005-01-21 | 2006-01-17 | シグナル産生成分として使用する組成物及びこれを用いる方法 |
EP06718583A EP1856529B1 (en) | 2005-01-21 | 2006-01-17 | Compositions for use as a signal generation component and methods of using same |
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Cited By (5)
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US20100233734A1 (en) * | 2009-03-16 | 2010-09-16 | Roderick Nicholas Hobbs | Passivation of surfaces after ligand coupling |
US8329417B2 (en) | 2008-04-29 | 2012-12-11 | Psychemedics Corporation | Non-proteolytic method for the determination of analytes in keratinized structures |
EP2623987A1 (en) | 2008-04-29 | 2013-08-07 | Psychemedics Corporation | Solid phase multi-analyte assay |
WO2014005065A1 (en) | 2012-06-28 | 2014-01-03 | Psychemedics Corporation | Detection of analytes in hair wash samples |
US9146233B2 (en) | 2006-11-13 | 2015-09-29 | Perkinelmer Health Sciences, Inc. | Detecting molecular interactions by fluorescence resonance energy transfer on a solid-phase support |
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US9347947B2 (en) | 2009-03-12 | 2016-05-24 | Siemens Healthcare Diagnostics Inc. | Immunoassays employing non-particulate chemiluminescent reagent |
US8153442B2 (en) * | 2009-10-21 | 2012-04-10 | Siemens Healthcare Diagnostics Inc. | Stabilization of signal generation in particles used in assays |
US10127349B2 (en) | 2010-07-16 | 2018-11-13 | Elitechgroup B.V. | Method of producing oligomer affinity pairs |
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US5466609A (en) * | 1990-10-31 | 1995-11-14 | Coulter Corporation | Biodegradable gelatin-aminodextran particle coatings of and processes for making same |
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- 2006-01-17 PT PT06718583T patent/PT1856529E/pt unknown
- 2006-01-17 ES ES06718583T patent/ES2392730T3/es active Active
- 2006-01-17 WO PCT/US2006/001529 patent/WO2006078618A2/en active Application Filing
- 2006-01-17 JP JP2007552204A patent/JP4741607B2/ja active Active
- 2006-08-04 US US11/499,519 patent/US7691647B2/en active Active
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Cited By (7)
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US9146233B2 (en) | 2006-11-13 | 2015-09-29 | Perkinelmer Health Sciences, Inc. | Detecting molecular interactions by fluorescence resonance energy transfer on a solid-phase support |
US8329417B2 (en) | 2008-04-29 | 2012-12-11 | Psychemedics Corporation | Non-proteolytic method for the determination of analytes in keratinized structures |
EP2623987A1 (en) | 2008-04-29 | 2013-08-07 | Psychemedics Corporation | Solid phase multi-analyte assay |
US10539580B2 (en) | 2008-04-29 | 2020-01-21 | Psychemedics Corporation | Solid phase multi-analyte assay |
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US20060166376A1 (en) | 2006-07-27 |
ES2392730T3 (es) | 2012-12-13 |
US20060270063A1 (en) | 2006-11-30 |
US7691647B2 (en) | 2010-04-06 |
PT1856529E (pt) | 2012-09-24 |
EP1856529A4 (en) | 2010-07-07 |
EP1856529A2 (en) | 2007-11-21 |
US20090325316A1 (en) | 2009-12-31 |
JP2008528969A (ja) | 2008-07-31 |
JP4741607B2 (ja) | 2011-08-03 |
WO2006078618A3 (en) | 2006-11-16 |
EP1856529B1 (en) | 2012-07-25 |
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